One goal of the Human Genome Initiative is to determine the 3 billion base pair genotype of Homo sapiens. The minimum number of templates required to sequence a human sized genome is 60 million using random shotgun cloning methods, with an equivalent number of associated sequencing reactions. The goal of this proposal is to develop an integrated cloning and sequencing system which will multiplex template purification three fold and DNA sequencing six fold over conventional methods. A new vector was developed to co-clone three DNA fragments in a single ligation reaction for isolation as a single template. Thus, one triplex template is equivalent to three conventional plasmid purifications. The six primer binding sites flanking each of the three cloned fragments permits six sets of DNA sequencing reactions from a single template. A simple strategy to multiplex the sequencing reactions improves the throughput of this step by six fold over conventional methods. The triplex cloning/sextaplex sequencing system is completely compatible with existing methods and instrumentation and does not require the development of specialized apparatus for successful implementation. PROPOSED COMMERCIAL APPLICATION: The triplex cloning/sextaplex sequencing system will provide the Human Genome Project with a simple method for reducing the number of template purifications three-old and DNA sequencing reactions six-old. This combinatorial cloning and sequencing method will result in significant savings in time, labor and expensive reagents, as well as improving the throughput of existing methods.